Die Entwicklung der NU-ELISA-Methode wurde von NIH RO1AG023687 unterstützt.

1. Mammalian Cell Culture NU-ELISA kann auf jedem Säugetier-Zelle Typ, der in Kultur gezüchtet werden können durchgeführt werden. Wir bevorzugen die Vorbereitung mäßige bis große Chargen von Zellen, so dass Nukleosomen in ausreichender Menge isoliert werden kann, um mehrere identisch geladen ELISA-Platten vorzubereiten, so dass Tests mit verschiedenen Antikörpern (ABS). Die folgenden Kultur Skalen bieten reichlich Material: Für Maus embyronic Stammzellen wachsen ein oder zwei 15 cm-Platten von Zellen ohne Feeder-Zellen. Für Fibroblasten wachsen 5 bis 10 15 cm-Platten bis zur Konfluenz. 2. Isolierung der Zellkerne Hinweis: Alle Schritte werden auf Eis mit vorgekühlten Puffer, außer wie angegeben. <ol><li> Trypsinize Zellen mit 3 ml Trypsin pro Platte, kombinieren und mit 20 ml eiskaltem PBS / Butyrat. Zentrifugation bei 1000 rpm für 5 min. </li><li> Resuspendieren der Zellen in 10 ml PBS / Butyrat und Zentrifuge bei 1000 rpm für 5 min. </li><li> Resuspendieren in 4 ml Lysepuffer mit Protease-Inhibitoren (Sigma-Aldrich P-8340). Dounce homogenisieren 20 Hübe mit Typ-B Pistill auf Eis. </li><li> Zentrifugation bei 2000g für 10 min bei 4 ° C. (Der Überstand enthält Zytoplasma, die nicht für dieses Protokoll benötigt wird, aber es kann für andere Zwecke gespeichert werden, wenn gewünscht). </li><li> Das Pellet in 2 ml eiskaltem Waschpuffer C (mit Protease-Inhibitoren). </li><li> Schicht der resuspendierten Material über 5 ml 30% Saccharose-Kissen, dann auf 2400g zentrifugieren Sie für 5 min in einem Ausschwingrotor. Kerne werden durch Kissen zu migrieren, und Schutt bleibt an der Oberfläche. </li><li> Entfernen Sie alle Flüssigkeitsvolumen sorgfältig und resuspendieren die Kerne in 250 ul eiskaltem Waschpuffer C + Protease-Inhibitoren. </li></ol> 3. Isolierung von Nukleosomen durch in situ Mikrokokken-Nuclease (MNase) Verdauung Wir verwenden ein Verfahren, bei dem Chromatin in situ innerhalb Kerne ist durch Infusion von ihnen mit MNase, gefolgt von einer hypotonen Behandlung kostenlos erhalten Nukleosomen in den Überstand Laufwerk verdaut. <ol><li> Add 3 ul 0,1 M CaCl 2 auf die Probe. Legen Sie in 37 ° C Wärme zu blockieren. Lassen Sie die 37 ° C Temperatur annehmen. </li><li> Add 2 Einheiten MNase (Mikrokokken-Nuclease, Sigma-Aldrich, bei 2 units/10μl in MNase Puffer gelöst), dann bei 37 ° C für 12 min, mit häufigem Mischen mit einer Pipettenspitze. </li><li> Add 6 ul von 0,5 M Na-EDTA, pH 8,0, um die Reaktion zu stoppen. Auf Eis gelegt. </li><li> Zentrifugation bei 2000g für 4 min, Überstand verwerfen. Das Pellet in 300 ul 0,2 mM Na-EDTA. Auf Eis für 1 h mit gelegentlichen sanften Pipettieren. (Diese hypotonen Bedingungen befreien freien Nukleosomen aus Kernen). </li><li> Zentrifugation bei 3000 g für 4 min bei 4 ° C. Speichern der Überstand, der freien Nukleosomen enthält, auf dem Eis. </li><li> Das Pellet erneut mit 300 ul 0,2 mM Na-EDTA. Auf Eis für 1 h mit gelegentlichen sanften Pipettieren. </li><li> Zentrifugation bei 3000g für 4min bei 4 ° C. Behalten Sie den Überstand und verbinden sich mit dem ersten Überstand aus Schritt 5. Die daraus resultierende mononucleosome Präparate können aliquotiert und bei -80 ° C gelagert werden </li></ol> Hinweis: Die Höhe des Chromatins grob durch Messung der Absorption bei 260 nm einer 10 ul Probe quantifiziert werden hinzugefügt bis 990 l Wasser. A 260 = 10 (bereinigt um die 1 / 100 Verdünnung) entspricht etwa 1mg/ml von Chromatin. Auch während der oben genannten Verfahren können kleine Proben aufbewahrt und später analysiert für ihre DNA-Gehalt, um die Qualität und den Umfang der MNase Aufschlüsse, die überwiegend enthalten, sollten DNA von 146 bp in der Länge zu überwachen. Laddering ist bezeichnend für unvollständige MNase Verdauung. Hinweis: Abb.1 enthält eine Zusammenfassung der diagrammed nuklearen Isolation und MNase Aufschlüsse Schritte. 4. Nucleosome-ELISA (NU-ELISA) Wir erkennen PTMs auf Fraktionen, die nativen intakten nuclesosomes, die auf 96-Well-ELISA Mikrotiterplatten immobilisiert wurden. Für jede Probe, wir machen eine Reihe von 2-fach Verdünnungen, und diese sind auf Brunnen in dreifacher Ausfertigung beschichtet. <ol><li> Coat MaxiSorp Platten über Nacht bei 4 ° C mit 50 ul / well der Nukleosomen in Beschichtungs-Puffer verdünnt. Empfohlene serielle zweifache Verdünnungen der Nukleosomen sind aus oberen Reihe auf der unteren Reihe durch Zugabe von 0,1 pg, 0,05 pg, 0,025 ug, 0.0125 ug 0,00625 ug 0,00313 ug 0,00156 pg vorbereitet, mit Beschichtungs-Puffer nur (0 pg Chromatin) im unteren Reihe. (Hinweis: Platte Abdeckungen sind für diesen Schritt erforderlich.) </li><li> Am Morgen waschen Sie die Platten 4 mal mit 200 ul / well PBS/0.5% Tween-20 bei Raumtemperatur (RT) für eine Summe von 10 min mit einer Platte Scheibe. </li><li> Block 1 h bei RT mit 100 ul / well PBS/0.05% Tween-20 / 5% BSA. </li><li> Entfernen Sie die Blocking-Puffer durch Schütteln des invertierten Platte zügigüber ein Waschbecken. Cover und speichern Sie die Platten bei -20 ° C, oder gehen Sie direkt zum nächsten Schritt. </li><li> Add 1 ° Abs in einem Volumen von 50 ul / well verdünnt 1:1000 (oder je nach Bedarf) in PBS/0.05% Tween-20 / 5% BSA bei RT für 1 Stunde auf einem Rotator inkubiert. </li><li> Wash Platten 4 mal mit 200 ul / well PBS/0.5% Tween-20 bei RT und für eine Summe von 10 min in einem Plattenwäscher. </li><li> Add Meerrettichperoxidase (HRP)-konjugiertem 2 ° Abs, verdünnt 1:5000 (oder je nach Bedarf) in PBS/0.05% Tween-20 / 5% BSA bei RT für eine Stunde auf einem Rotator. </li><li> Wash Platten 4 mal mit 200 ul / well PBS/0.5% Tween-20. Jeder waschen bei RT und für insgesamt 10 Minuten mit einem Plattenwäscher. </li><li> Entwickeln Sie die Platten durch Zugabe von 50 ul TMB Substrat in jede Vertiefung für 10 min bei RT. Stoppen der Reaktion durch Zugabe von 50 ul / Vertiefung 2N H 2 SO 4 und lesen Sie die Platten 450 nm. (Tipp: Zentrifugation bei 1500 rpm für 2 min mit einem Platten-Rotor, um die Luftblasen in den Vertiefungen vor dem Lesen Extinktionen abführen). </li><li> Export der Messwerte in Excel Dateien für quantitative und statistische Analysen. </li></ol> Reagenzien <table border="1"><tbody><tr><td> PBS / Butyrat </td></tr><tr><td> 135 mM NaCl </td></tr><tr><td> 2,5 mM KCl </td></tr><tr><td> 8 mM Na 2 HPO 4 </td></tr><tr><td> 1,5 mM KH 2 PO 4 </td></tr><tr><td> 10 mM Na-Butyrat </td></tr></tbody></table><table border="1"><tbody><tr><td> Lysispuffer </td></tr><tr><td> 250 mM Saccharose </td></tr><tr><td> 10 mM Tris-HCl, pH 7,4 </td></tr><tr><td> 10 mM Na-Butyrat </td></tr><tr><td> 4 mM MgCl 2 </td></tr><tr><td> 0,1% Triton X-100 </td></tr></tbody></table><table border="1"><tbody><tr><td> Waschpuffer C </td></tr><tr><td> 250 mM Saccharose </td></tr><tr><td> 10 mM Tris-HCl, pH 7,4 </td></tr><tr><td> 10 mM Na-Butyrat </td></tr><tr><td> 4 mM MgCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Saccharose-Kissen </td></tr><tr><td> 30% (w / v) Saccharose in Waschpuffer C </td></tr></tbody></table><table border="1"><tbody><tr><td> Microccocal Nuclease Puffer </td></tr><tr><td> 5 mM NaPO 4, pH 7,0 </td></tr><tr><td> 0,025 mm CaCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Coating-Puffer </td></tr><tr><td> 80ml Lösung A + 170ml Lösung B + 250ml dH 2 O </td></tr><tr><td> Lösung A: 0,2 M Na 2 CO 3 </td></tr><tr><td> Lösung B: 0,2 M NaHCO 3 </td></tr></tbody></table> 5. Repräsentative Ergebnisse Mit dem NU-ELISA-Methode, eine Reihe von mehreren identischen Platten hergestellt werden, die beladen mit Chromatin in Form von mononucleosomes vorbereitet werden können. Wir bereiten in der Regel Folge von identisch beladenen Platten, so dass jeder mit unterschiedlichen anti-PTM-spezifischen Antikörpern (Abs) untersucht werden können. Wir haben auch stets einen Teller, der mit einem Ab, dass Histone erkennt ohne Rücksicht auf deren Modifikation Staat insgesamt Chromatin Ladekontrolle untersucht werden können. Diese Kontrolle ist notwendig, um später quantitativ zu vergleichen PTM Inhalt der Nukleosomen aus verschiedenen Proben hergestellt von wesentlicher Bedeutung. Um quantifizieren das Niveau der PTMs innerhalb einer bestimmten Chromatin Probe korrigieren wir die Rohstoffe Extinktionswerte mit diesem Ansatz: Zuerst subtrahieren wir alle Hintergrund-Signal mit Werten aus Kontrollvertiefungen enthält keine Chromatin (Hintergrund ist in der Regel vernachlässigbar) erhalten. Wir haben dann zu standardisieren PTM-spezifische Signale zu NU-ELISA Ergebnisse aus einem identisch geladen Platte, die mit einem Ab, dass Nukleosomen unabhängig von deren Modifikation Staat erkennt untersucht wurde erhalten. (Wir haben polyklonalen Abs spezifisch für Histone H2A H2B oder für diesen Zweck verwendet). Wir bestimmen dann Mittelwerte und Varianzen für jede Verdünnung von Chromatin, und verwenden Sie Daten aus dem linearen Teil der ELISA-Test erhalten. Ausführliche Beispiele von NU-ELISA mathematischen und statistischen Analysen wurden bisher 4 gemeldet <img src="/files/ftp_upload/2593/2593fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Schematische diagam der NU-ELISA Schritte. A. Mammalian Cells geerntet werden; B. Kerne sind aus Zellen, die durch Dounce h getrenntomogenization, C. aufgeschlossenen Zellen auf der Spitze eines 30% w / v Saccharose-Kissen, D. Nach dem Zentrifugieren geladen werden, werden Kerne in das Pellet abgeschieden; E, F. Chromatin sind in situ überwiegend mononucleomes durch MNase verdaut; G, H , Ich. Löslich mononucleosomes durch hypotone Behandlung und wiederholte Zentrifugation des restlichen Kernmaterial extrahiert. J. Mononucleosomes aus verschiedenen Proben (mit samp. 1 und 2 in diesem Fall) auf Mikrotitervertiefungen in Serie identisch beladenen Platten beschichtet und verhört mit PTM-Risikoabdeckung spezieller ABS-und PTM-unabhängige Abs insgesamt Chromatin Laden zu bestimmen. K. Darstellung der Differenz-Signal-Erkennung Ebenen in zwei Proben, die in ihrer PTM Inhalt unterscheiden, aber ähnliche insgesamt Chromatingehalt, wie anti-H2B-Immunreaktivität beurteilt.

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+and+an+epigenetic+code.">Histone acetylation and an epigenetic code.</a> Bioessays. 22, 836-845 (2000).</li><li>Dai, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epiproteomic+signatures+distinguish+embryonic+stem+cells+from+differentiated+cells.">Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.</a> Stem Cells. 25, 2567-2574 (2007).</li><li>Tanasijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+accumulation+of+epigen+etic+heterogeneity+during+human+ES+cell+culture.">Progressive accumulation of epigen etic heterogeneity during human ES cell culture.</a> Epigenetics. 4, 330-338 (2009).</li><li>Thomas, J. O., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+fractionation+of+chromatin+and+linker+histones.">Isolation and fractionation of chromatin and linker histones.</a> Chromatin: a practical approach. , 12-14 (1998).</li><li>Thorne, A. W., Cary, P. D., Crane-Robinson, C., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+separation+of+core+histones+and+non-histone+chromosomal+proteins.">Extraction and separation of core histones and non-histone chromosomal proteins.</a> Chromatin: a practical approach. , 38-39 (1998).</li></ol>

NU-ELISA bietet eine Methode, um den globalen Status der Nukleosomen PTMs Gegenwart in einem bestimmten Zelltyp zu ermitteln. NU-ELISA-Studien haben gezeigt, dass die globale nucleosome Änderung Staaten Vergleiche unterschiedlicher Zelltypen 4 unterscheiden. Darüber hinaus ändern NU-ELISA PTM-Profilen, wenn Zellen epigenetische modulatorische Substanzen wie ausgesetzt sind Trichostatin-A oder, wenn die Maus embyronic Stammzellen sind 4 differenziert. Die Methode wurde auch erfolgreich auf das Chromatin der menschlichen ES-Zellen 5-Studie angewandt. Es ist wichtig zu beachten, die NU-ELISA, in seiner jetzigen Form kann nur der Composite-Signatur PTMs innerhalb des Summe der zellulären Epigenom zu erkennen. Dies unterscheidet sich deutlich von genomweiten Chromatinimmunpräzipitation, der die Verteilung eines bestimmten PTM über spezifische genetische Loci bestimmen kann. Die ersten Schritte des NU-ELISA-Verfahren werden aus den bisherigen Methoden angepasst mononucleosomes aus Säugetier-Kerne 6,7 isolieren. Diese angepasste Methoden bieten eine sinnvolle Möglichkeit, qualitativ hochwertige intakt mononucleosomes aus Säugetier-Zellen zu erhalten, und die erhaltenen Fraktionen sind umfassend in ihrem Chromatin Inhalt, sondern enthalten eine Vielzahl von zusätzlichen Kernmaterial. Da die spezifischen Abs verwendet werden, ist verunreinigt nicht nukleosomalen Material gut in die NU-ELISA-Test vertragen, im Gegensatz zu Massen-spec Ansätze, die eine größere Reinheit erfordern. Allerdings ist NU-ELISA empfindlicher und quantitative als Western-Blot-4, so dass eine gute Alternative zu Western und Massenspektrometrie zum Nachweis von nukleosomalen PTMs.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> Mikrokokken Nucleae </td><td> Sigma </td><td> N3755 </td><td> Machen Aliquots von 2 U/10μl Puffer bei -20 ° C gelagert </td></tr><tr><td> Nunc MaxiSorp Mikrotiterplatten </td><td> Fischer </td><td> 12-565-135 </td><td> Klebeplatte Abdeckungen können auch durch Fisher bestellt werden </td></tr><tr><td> Automatisierte Plattenwäscher </td><td></td><td></td><td></td></tr><tr><td> 1-Step TMB-Substrat ELISA </td><td> Durchstechen </td><td> 34028 </td><td> Lagerung bei 4 ° C vorwärmen auf RT vor dem Gebrauch oder als auf dem Datenblatt gerichtet </td></tr><tr><td> Mikrotiterplatten-Reader </td><td> Bio-Rad-Mikroplatten-Reader (Modell: 680) </td><td></td><td> Die meisten kolorimetrischen Plattenlesegerät Modelle eignen sich </td></tr></tbody></table>

detection of post-translational modifications on native intact nucleosomes by elisa

Das Genom der Eukaryoten existiert als Chromatin, die sowohl DNA und Proteine ​​enthält. Die Grundeinheit des Chromatins ist das Nukleosomen, die 146 Basenpaaren der DNA mit je zwei der Histone H2A, H2B, H3 und H4 1 zugeordnet enthält. Die N-terminalen Enden der Histone sind reich an Lysin und Arginin und post-transkriptionell durch Acetylierung, Methylierung und andere post-translationale Modifikationen (PTM) modifiziert. Die PTM-Konfiguration der Nukleosomen kann sich auf die transkriptionelle Aktivität von assoziierten DNA, wodurch eine Art der Genregulation, dass epigenetische in der Natur ist 2,3. Wir entwickelten eine Methode namens Nukleosomen ELISA (NU-ELISA) zur quantitativen Bestimmung globaler PTM Unterschriften von Nukleosomen aus Zellen extrahiert. NU-ELISA ist empfindlicher und quantitative als Western-Blot, und ist nützlich, um die epiproteomic Zustand der spezifischen Zelltypen zu verhören. Dieses Video Zeitschriftenartikel zeigt detaillierte Verfahren zur NU-ELISA-Analyse durchzuführen.

Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Nucleosome ELISA (NU-ELISA) ist eine sensible und quantitative Methode zur globalen Muster der post-translationale Modifikationen in Zubereitungen native, intakte Nukleosomen zu erkennen. Diese Änderungen umfassen Methylierungen, Acetylierungen und Phosphorylierungen an bestimmten Histon Aminosäurereste und damit NU-ELISA bietet eine globale Proteom-Test des Gesamtsystems Chromatin Modifikation Staaten von spezifischen Zelltypen.

Erkennung von Post-translationale Modifikationen auf Native Intact Nukleosomen mittels ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

detection-post-translational-modifications-on-native-intact

Research

JoVE Journal

Biology

17.2K Aufrufe.  Stanford University. Nucleosome ELISA (NU-ELISA) ist eine sensible und quantitative Methode zur globalen Muster der post-translationale Modifikationen in Zubereitungen native, intakte Nukleosomen zu erkennen. Diese Änderungen umfassen Methylierungen, Acetylierungen und Phosphorylierungen an bestimmten Histon Aminosäurereste und damit NU-ELISA bietet eine globale Proteom-Test des Gesamtsystems Chromatin Modifikation Staaten von spezifischen Zelltypen.

Serie: Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Erkennung von Functional Matrix-Metalloproteinasen durch Zymographie

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Forschung

Biologie

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Die Analyse der Funktion von kleinen GTPasen durch Mikroinjektion von Plasmiden in polarisierten Epithelzellen

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Erkennung von Histon-Modifikationen in Pflanzenblättern

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Bäckerhefe Protein Extraktion und Reinigung für das Studium der Funktion, Wechselwirkungen, und post-translationale Modifikationen

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Bezeichnung des Post-translationale Modifikationen von Pflanzenproteinkomplexe

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Isolierung von spezifischen genomischen Regionen und Identifizierung von assoziierten Moleküle durch enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En Gesichtserkennung von Stickstoffmonoxid und Superoxid in Endothelschicht Intact Arterien

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

High-throughput CRISPR Vector Konstruktion und Charakterisierung von DNA-Modifikationen durch Erzeugung von Tomaten Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Komplette Workflow zur Analyse von Histon-post-translationale Modifikationen Mit Bottom-up-Massenspektrometrie: Von Histone Extraktion in Datenanalyse

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Vergleich von Tabakwirtszellprotein Abbaumethoden von Intact Pflanzen oder Blanchieren durch Wärmebehandlung von Extrakten

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...